The invention relates to a protein for regulating apoptosis and to a nucleic acid encoding the protein. The protein and nucleic acids are useful in the regulation of apoptosis, and in methods for diagnosing or detecting apoptosis. The invention also relates to antibodies directed against the protein.
Apoptosis is a type of cell death, which is thought to be genetically programmed, and which is distinct from the process of necrosis. During apoptosis, cells generally se their cell junctions and microvilli, the cytoplasm condenses and nuclear chromatin marginates into a number of discrete masses. Cory, S. Nature 367:317-318 (1994).
Apoptosis can be induced via a variety of receptors, commonly referred to as xe2x80x9cdeath receptors.xe2x80x9d These receptors contain a xe2x80x9cdeath domainxe2x80x9d (DD). Examples include CD95, TNF-RI, DR3, DR4 or DR5, which induce apoptosis signal paths after binding their respective ligands. For example, after the CD95 ligand binds to the CD95 receptor, the receptor interacts with the adapter protein FADD/MORTI to induce the recruitment and activation of the protease FLICE/Caspase-8 at the DISC xe2x80x9cdeath inducing signaling complex.xe2x80x9d FADD and FLICE contain xe2x80x9cdeath effector domains (DED).
Apoptosis can be inhibited by the transcription of anti-apoptotic genes, i.e. by the gene products thereof. For example, the protein FLIP xe2x80x9cFLICE-inhibitory proteinxe2x80x9d inhibits the CD95 apoptosis signal path. German patent 19713434 of Deutsches Krebsforschungszentrum (the German Cancer Research Center). There is a need in the art for novel compounds for regulating of apoptosis, and for methods for using such compounds. It is thus an object of the present invention to provide such compounds and methods.
The invention relates generally to proteins for regulating and/or inducing apoptosis. The proteins of the invention generally include the amino acid sequence of FIG. 1A (SEQ ID NO: 1) or an amino acid sequence which is substantially similar to the amino acid sequence of FIG. 1A (SEQ ID NO: 1). Alternatively, the protein may consist of or consist essentially of the amino acid sequence of FIG. 1A (SEQ ID NO: 1) or an amino acid sequence which is substantially similar to the amino acid sequence of FIG. 1A (SEQ ID NO: 1).
In one aspect the invention relates to an amino acid sequence differing from the amino acid sequence of FIG. 1A (SEQ ID NO: 1) by one or more amino acids, wherein the DNA encoding the amino acid sequence hybridizes with the DNA encoding the amino acid sequence of FIG. 1A (SEQ ID NO: 1). Preferably, the amino acid sequence differs from the amino acid sequence of FIG. 1A (SEQ ID NO: 1) by no more than 1, 2, 3, 4, or 5 amino acids. The amino acid sequence may also be an active fragment of the sequence of FIG. 1A (SEQ ID NO: 1) or of an amino acid sequence which is substantially similar to an active fragment. Examples of active fragments include amino acids 1-114 of FIG. 1A (SEQ ID NO: 1) or amino acids 109-318 of FIG. 1A (SEQ ID NO: 1). Additionally, the amino acid sequence may be a truncation of the amino acid sequence of FIG. 1A (SEQ ID NO: 1), i.e., truncated at the C-terminus and/or the N-terminus, preferably by no more than 1, 2, 3, 4, or 5 amino acids at either terminus. The amino acid sequence may also comprise one or more conservative substitutions.
In a related aspect, the amino acid sequence differs from the amino acid sequence of FIG. 1A (SEQ ID NO: 1) by one or more amino acids and hybridizes to the DNA encoding the amino acid sequence of FIG. 1A (SEQ ID NO: 1), preferably under stringent conditions.
The protein of the invention is suitably provided as a component of a pharmaceutical composition.
The invention also relates to a nucleic acid, such as a DNA or RNA encoding any of the proteins of the invention. In one aspect, the DNA comprises the nucleotide sequence of FIG. 1A (SEQ ID NO: 2) or a nucleotide sequence which is substantially similar to the nucleotide sequence of FIG. 1A (SEQ ID NO: 2). The DNA may differ from the DNA of FIG. 1A (SEQ ID NO: 2) by one or more degenerate codons. In a preferred aspect, the DNA comprises a nucleotide sequence corresponding to, or substantially similar to, nucleotides 28 to 369 of FIG. 1A (SEQ ID NO: 2), as provided in SEQ ID NO: 7, nucleotides 352 to 981 of FIG. 1A (SEQ ID NO: 2), as provided in SEQ ID NO: 8, or the nucleotide sequence of FIG. 1B (SEQ ID NO: 4). The nucleotide sequence is suitably provided as a component of an expression plasmid or an expression cassette, or as a component of a cell transformed by a nucleic acid comprising the nucleotide sequence.
The invention also relates to a process for preparing a protein of the invention. In one aspect, the method of production comprises culturing a cell transformed by an expression plasmid comprising a nucleotide sequence selected from the following: (a) the nucleotide sequence of FIG. 1A (SEQ ID NO: 2); (b) a nucleotide sequence which is substantially similar to the nucleotide sequence of FIG. 1A (SEQ ID NO: 2); (c) a nucleotide sequence which is substantially similar to the nucleotide sequence of FIG. 1A (SEQ ID NO: 2); (d) a nucleotide sequence which hybridizes to the nucleotide sequence of FIG. 1A (SEQ ID NO: 2), preferably under stringent conditions; (e) a nucleotide sequence which differs from the nucleotide sequence of FIG. 1A (SEQ ID NO: 2) by one or more degenerate codons; (f) a nucleotide sequence corresponding to nucleotides 28 to 369 of FIG. 1A (SEQ ID NO: 2), as provided in SEQ ID NO: 7, or a nucleotide sequence substantially similar thereto; (g) a nucleotide sequence corresponding to nucleotides 352 to 981 of FIG. 1A (SEQ ID NO: 2), as provided in SEQ ID NO: 8; and (h) the nucleotide sequence of FIG. 1B (SEQ ID NO: 4) or a nucleotide sequence substantially similar thereto.
In another aspect, the invention provides an antibody which binds, preferably which specifically binds, to a protein of the invention. The antibody is preferably a monoclonal antibody. In a related aspect, the invention relates to a hybridoma cell line expressing an antibody of the invention.
The invention also relates to a method for detecting in a sample the presence a protein of the invention. In general, the method employs the following steps: (a) contacting the sample with a an antibody which specifically binds to a target protein of the invention; and (b) analyzing the sample for the presence of antibody specifically bound to target protein.
Similarly, the invention relates to a method for islolating a protein of the invention. The method generally employs the following steps: (a) contacting a composition comprising the target protein with an antibody which specifically binds to the target protein; (b) eluting the target protein from the antibody. The eluted protein may then be separated using known procedures, for example, standard cross-flow filtration techniques known in the art.
In another aspect, the invention relates to a method for inducing apoptosis in a cell. In this method, apoptosis is induced by contacting the cell with a protein of the invention. The cell may be contacted in vivo by administration of the protein of the invention to a subject in an amount sufficient to induce apoptosis. The method is useful, for example, in the treatment of diseases of the immune system and in the treatment of neoplasms. The administration may be direct, by administering the protein to the subject, or indirect, by administering to the subject a nucleotide encoding the protein for expression in the subject.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. For purposes of the present invention, the following terms are defined below.
The term xe2x80x9cidenticalxe2x80x9d or xe2x80x9cidentityxe2x80x9d in the context of two nucleic acids or polypeptide sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection.
The term xe2x80x9csubstantial identityxe2x80x9d or xe2x80x9csubstantial similarityxe2x80x9d in the context of a polypeptide indicates that a polypeptide comprises a sequence with at least 70% sequence identity to a reference sequence, or preferably 80%, or more preferably 85% sequence identity to the reference sequence, or most preferably 90% identity over a comparison window of about 10-20 amino acid residues. An indication that two polypeptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a polypeptide is substantially identical to a second polypeptide, for example, where the two peptides differ only by a conservative substitution.
An indication that two nucleotide sequences are substantially identical is that the polypeptide which the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
Another indication that two nucleotide sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. Stringent conditions are sequence dependent and are different under different environmental parameters. Generally, stringent conditions are selected to be about 5xc2x0 C. to 20xc2x0 C. lower than the thermal melting point (T[m]) for the specific sequence at a defined ionic strength and pH. The T[m] is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. However, nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
The phrases xe2x80x9cspecifically binds to a proteinxe2x80x9d or xe2x80x9cspecifically hybridizes toxe2x80x9d or xe2x80x9cspecifically immunoreactive with,xe2x80x9d refers to a binding reaction which is determinative of the presence of the protein in the presence of a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind preferentially to a particular protein and do not bind in a significant amount to other proteins present in the sample. Specific binding to a protein under such conditions requires an antibody that is selected for its specificity for a particular protein. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, N.Y., for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
The terms xe2x80x9cisolated,xe2x80x9d xe2x80x9cpurified,xe2x80x9d or xe2x80x9cbiologically purexe2x80x9d refer to material which is substantially or essentially free from components which normally accompany it as found in its native state.
The term xe2x80x9cantibodyxe2x80x9d as used herein, includes various forms of modified or altered antibodies, such as an intact immunoglobulin, various fragments such as an Fv fragment, an Fv fragment containing only the light and heavy chain variable regions, an Fv fragment linked by a disulfide bond (Brinkmann, et al. Proc. Natl. Acad. Sci. USA, 90: 547-551 (1993)), a Fab or (Fab)""2 fragment containing the variable regions and parts of the constant regions, a single-chain antibody and the like (Bird et al., Science 242: 424-426 (1988); Huston et al., Proc. Nat. Acad. Sci. USA 85: 5879-5883 (1988)). The antibody may be of animal (especially mouse or rat) or human origin or may be chimeric (Morrison et al., Proc Nat. Acad. Sci. USA 81: 6851-6855 (1984)) or humanized (Jones et al., Nature 321: 522-525 (1986), and published UK patent application #8707252).
The terms xe2x80x9cpeptide,xe2x80x9d xe2x80x9cpolypeptidexe2x80x9d and xe2x80x9cproteinxe2x80x9d are used interchangeably herein to refer to an amino acid chain with two or more amino acid residues, and also includes branched and circularized amino acid chains.